Theses and Dissertations from UMD
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Item SURVIVAL AND PERSISTENCE OF LISTERIA AND ESCHERICHIA COLI AND CHANGES IN PHYSICOCHEMICAL PARAMETERS IN AQUAPONICS SYSTEMS DURING LETTUCE PRODUCTION(2023) ALHAMMAD, GHADAH ABDULRAHMAN; Lei, David K. Y.; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Aquaponic (AP) food production systems integrate aquaculture and hydroponics in soilless controlled environments while conserving space and natural resources (soil, water, air). Increasing global demand for high-quality quality nutritious, safe food continues to increase exponentially. Information specific to AP is needed to validate operational and handling practices for AP food safety. The first study is to assess the persistence of E. coli TVS 354 in bench-scale aquaponic systems (180 days after primary plant harvest). Methods: follow-up on the previous study and evaluate growth and persistence of E. coli TVS 354 at 180 days after primary plant harvest. Samples were collected from 10 independent aquaponic systems and bacterial growth was evaluated including aerobic plate count (APC), coliform bacteria (CB), heterotrophic plate count (HPC), and cultural enrichment for E. coli TVS 354 levels. Results: E. coli TVS 354 was not detected in samples from hydrotons (25 g), filters, fish feces, core and roots. The aerobic bacteria count of fish and plant tank samples ranged between 2.5 to 3 log CFU/mL for all treatments, while aerobic bacteria count on the core of the lettuce plant was significantly lower, ranging from 1.44 to 2.08 log CFU/mL. The second study is to evaluate the survival of Listeria innocua 2066 in effluent from fish tanks and plant tanks of aquaponic research units. Methods: four different treatments: fish tank water, fish tank water-replenished, plant tank water, and plant tank water-replenished. Each bottle was inoculated with Listeria innocua 2066, and bacterial growth and water chemistry conditions were evaluated over 7 days to identify physiochemical parameters associated with Listeria innocua 2066 survival and growth. Results: In 24 hours a significant reduction (p<0.05) in Listeria inccoua 2066 populations was observed for all treatments with a total reduction 3.9-3.7 log CFU/mL. Listeria innocua 2066 counts were lowest on day 7 for treatment groups, ranging from 1.1 log CFU/mL to 1.0 log CFU/mL. Treatments that were replenished daily from the aquaponic systems had higher counts of Listeria innocua 2066 compared to treatments that were non-replenished. The lowest APC on day 0, immediately after inoculation, ranged from 4.16 log CFU/mL to 4.32 log CFU/mL. A significant increase (P<0.05) in APC count was observed for all treatments in 24 hours while there is no significant difference in APC values between the treatments (p=0.35). The final and third study is to evaluate survival of inoculated Listeria innocua 2066 and nonpathogenic Escherichia coli and changes in AP physicochemical parameters. Methods: A four-week bench-scale AP experiment (n=12) with four goldfish (Carassius auratus)/aerated 37L tank, a 3-step biofilter, and four lettuce plants (Lactuca sativa var. Truchas)/37L hydroponic tank was conducted. Treatments were high and low-dose (6-log and 2-log CFU/mL, respectively) of Listeria innocua 2066-Er or E. coli-Rifr TVS 354, and uninoculated controls. Listeria innocua-2066-Er and E. coli-Rifr populations, mesophilic counts (APC), and physicochemical parameters (pH, temperature, dissolved oxygen, turbidity, ammonia, nitrite, and nitrate) were analyzed in plant tank water, and biofilters until plant harvest. Listeria innocua 2066-Er and E.coli-Rifr enumeration and APC (Petrifilm®) from lettuce shoots, roots and rockwool were determined at harvest. Results: Listeria innocua 2066 and E. coli TVS 354 populations declined significantly within 24 hours post-inoculation and were undetectable at day 14 and day 12 respectively (P <0.05). This decline was observed for both high and low-dose treatments. Listeria innocua 2066 and E. coli TVS 354 were detected in biofilters until week 4. At harvest, Listeria innocua 2066 and E. coli TVS 354 were recovered from lettuce roots, and rockwool, but not from plant leaves, and 100mL plant tank water grab samples. Lower leaves preharvest had a significantly higher APC (5.1- 6.4 log CFU/mL) relative to harvested lettuce upper leaves (2.8 - 4.2 log CFU/mL). Plant tank water pH had a significant effect (P<0.05) on Listeria innocua 2066 and E. coli TVS 354 survival. Significance: These results provide insights into the survival dynamics and sites of E. coli TVS 354 and Listeria innocua 2066 in AP and associated physicochemical conditions. These findings contribute to our understanding of potential food safety risks and associated risk factors such as inputs, physicochemical factors, and other environmental conditions in aquaponics systems.Item A SYNTHETIC TMRNA PLATFORM FOR ELUCIDATION OF BACTERIAL PROTEOME REMODELING UNDER STRESS(2022) Turner, Randi; McIver, Kevin; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Translational reprogramming is a key component of the bacterial stress response and is a function of mRNA stability, protein turnover and proteolysis. Total proteome measurements give a view of the stable proteome but can fail to capture dynamic changes under stress, including incomplete polypeptides that result from cleaved mRNAs or stalled translation events. Bacteria employ a nearly ubiquitous native ribosome rescue system, transfer-messenger RNA (tmRNA), that rapidly resolves stalled translational complexes and tags the incomplete polypeptides for degradation. Characterization of these tmRNA-tagged polypeptides could reveal previously unknown aspects of the bacterial stress response. To address this information gap, we have developed a synthetic tmRNA platform that reprograms the native system to allow for co-translational labeling of the incomplete polypeptides in live bacteria. A short tag reading frame (TRF) encoded on native tmRNA facilitates the addition of a natural peptidyl degradation tag to the polypeptides, and therefore offers an attractive modular domain to introduce synthetic peptide tag sequences and study the “degradome”. To study translational remodeling under stress, we modified the native tmRNA with an 6x-HIS isolation tag with the specific purpose of stabilizing, isolating, and characterizing the degradome in Escherichia coli. Using our inducible system, we have successfully isolated 6xHis-tagged proteins, verified dynamic controlled tagging, assessed broad-spectrum tag introduction with mass spectrometry. Our results capture known tmRNA substrates and excitingly show that tagged protein profiles are markedly different under stress. We investigated the shifting degradome in cells experiencing translational stress associated with serine starvation induced by serine hydroxamate. In cells lacking RelE, the mRNA interferease toxin that cleaves mRNA in the ribosome A site, we find a dramatic shift away from catalytic protein degradation and distinct, disparate enrichment of ribosomal proteins in the degradome under stress. These latter results suggest a new specific role for RelE in regulating ribosome protein abundance under translational stress conditionsItem Monitoring and Predicting the Microbial Water Quality in Irrigation Ponds(2022) Stocker, Matthew Daniel; Hill, Robert L; Environmental Science and Technology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Small- to medium-sized farm ponds are a popular source of irrigation water and provide a substantial volume of water for crop growth in the United States. The microbial quality of irrigation waters is assessed by measuring concentrations of the fecal indicator bacteria Escherichia coli (E. coli). Minimal guidance currently exists on the use of surface irrigation waters to minimize consumer health risks. The overall objective of this work was to provide science-based guidance for microbial water quality monitoring of irrigation ponds. Spatial and temporal patterns of E. coli were evaluated in two Maryland irrigation ponds over three years of observations. Patterns of E. coli were stable over the three years and found to be significantly correlated to patterns of water parameters such as temperature, dissolved oxygen, turbidity, and pH. The EPA Environmental Fluid Dynamics Code model was used to evaluate the spatial 3D heterogeneity of E. coli concentrations within the ponds. Significant differences in E. coli concentrations by sampling depth were found. Spatial heterogeneity of E. coli within the pond also resulted in substantial temporal variation at the irrigation pump, which was dependent on the intake location. Diurnal variation of E. coli concentrations was assessed for three farm ponds. E. coli concentrations declined from 9:00 to 15:00 for each pond, but statistically significant declines were only observed in two of the three ponds. Dissolved oxygen, pH, and electrical conductance were found to be the most influential environmental variables affecting E. coli concentrations. To better describe the relationships between E. coli and the environmental variables, four machine learning algorithms were used to estimate E. coli concentrations using water quality parameters as predictors. The random forest algorithm provided the highest predictive accuracy with R2 = 0.750 and R2 = 0.745 for Ponds 1 and 2, respectively, in the multi-year dataset containing 12 predictors. Temperature, electrical conductance, and organic matter content were identified as the most influential predictors. It is anticipated that the recommendations contained in this dissertation will be used to improve microbial monitoring strategies and protect public health.Item EVALUATION OF PUBLIC HEALTH RISK FOR ESCHERICHIA COLI O157:H7 IN CILANTRO(2019) Horr, Taryn; Pradhan, Albani; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The supply chain of cilantro was modeled for growth and die-off of Escherichia coli (E. coli) O157:H7 from infield and harvesting, transportation and storage and ultimately consumption at home. Using Visual Basic for Applications (VBA) macros and @RISK software, a simulation model was developed for exposure and estimation of illnesses. Test scenarios were modeled to determine the relative importance of different factors on the risk of illness. The developed model was simulated using Monte Carlo technique and Latin Hypercube sampling for 100,000 iterations. Results showed an increase in the mean E. coli O157:H7 concentration along the supply chain for cilantro grown in both winter and summer weather conditions. In the winter, the mean pathogen concentration increased from 5.6×10-5 CFU/g to 24.7 CFU/g from after harvest to after home storage, respectively. In summer conditions, the mean pathogen concentration increased from 3.2×10-4 CFU/g to 5.2×10-2 CFU/g. The inner quartile ranges (IQRs) for the same model conditions showed a decrease in E. coli O157:H7 concentration along the supply chain for cilantro grown in both winter and summer weather conditions. This indicates a majority of situations result in a decrease in E. coli O157:H7 concentration along the supply chain however rare situations can occur where the concentration will increase greatly. With a prevalence of 0.1% E. coli O157:H7 contamination for cilantro post-harvest used for illustration, the model predicted the mean number of illnesses per year due to the consumption of E. coli O157:H7 contaminated cilantro in the United States as 86 and 164 for cilantro grown during winter and summer conditions, respectively. Sensitivity analysis results indicated that transportation temperatures and quality of irrigation water had the largest impact on the number of illnesses per year. Scenario testing results for different risk factors demonstrated the importance of limiting and reducing cross contamination along the production chain, especially at higher initial prevalence levels and preventing temperature abuse during transportation from farm to retail, when reducing overall risk of illness. The developed risk model can be used to estimate the microbiological risks associated with E. coli O157:H7 in cilantro and determine areas along the supply chain with the most effect on the final concentration per serving for future mitigation strategies.Item Release, Survival, And Removal of Bovine Manure-Borne Indicator Bacteria Under Simulated Rainfall(2017) Stocker, Matthew Daniel; Hill, Robert L; Environmental Science and Technology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The effects of simulated rainfall intensities and its interactions with manure consistency and weathering on the release, survival, and removal of fecal indicator bacteria, Escherichia coli and enterococci, from land-applied dairy manure were evaluated. Rainfall intensity had significant effects on the number of bacteria in the soil following rainfall. Bacteria concentrations in soil decreased with increased soil depths and the topmost centimeter of soil accounted for the greatest proportion of bacteria. Escherichia coli persisted longer than enterococci once removed from manure. Manure consistency was not a significant factor in the removal of bacteria when manure was fresh, but as manure weathering progressed, consistency became a significant factor. The Vadas-Kleinman-Sharpley model was preferred over the exponential model for simulating the removal of manure-borne bacteria. Results of this work will be useful for improving predictions of the human health risks associated with manure-borne pathogenic microorganisms.Item Antimicrobial Resistance of Salmonella and E. coli from Pennsylvania Dairy Herds(2015) Cao, Huilin; Pradhan, Abani K.; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The emergence and dissemination of bacterial antimicrobial resistance has become a major public health concern. A total of 444 manure composite samples were collected from 80 dairy farms in Pennsylvania, representing pre-weaned calves, post-weaned calves, dry cows, and lactating cows. E. coli and Salmonella were isolated, and tested for antimicrobial susceptibility. Salmonella was isolated from at least one sample from 51 (64%) farms and was more prevalent in adult animals than young animals. The predominant serotypes were Cerro, Montevideo and Kentucky. Salmonella isolates were mostly susceptible to all antimicrobials. E. coli were commonly resistant to tetracycline, streptomycin, sulfisoxazole and ampicillin. Resistance of up to 8 classes of antibiotics was observed in E. coli isolated from young animals. The blaCMY- and blaCTX-M-carrying E. coli were detected in 35% and 5% of the farms, respectively. The presence of multi-drug resistant E. coli suggested potential risks to human health associated with dairy farming.Item Release and runoff/infiltration removal of Escherichia coli, enterococci, and total coliforms from land-applied dairy cattle manure(2014) Blaustein, Ryan Andrew; Hill, Robert L; Environmental Science and Technology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Simulating the rainfall-induced release of indicator bacteria from manure is essential to microbial fate and transport modeling with regard to water quality and food safety. Experiments were conducted to determine the effects of rainfall intensity, surface slope, and scale on the release of Escherichia coli, enterococci, and total coliforms from land-applied dairy manure. Rainfall intensity did not affect bacterial release dependencies on rainfall depth, but it did have a significant effect on the post-rainfall quantities of indicator bacteria in soil. While bacterial concentrations were evenly released into runoff and infiltration, the surface slope controlled the partitioning of total released bacterial loads. The proportion of E. coli released from manure exceeded enterococci, especially with infiltration flow. Scale had strong, inverse effects on the recovery of land-applied bacteria with runoff. These results will be used to improve microbial fate and transport models, critical for risk assessment of microbial contamination in the environment.Item Membrane models of E. coli containing cyclic moieties in the aliphatic lipid chain(2012) Pandit, Kunal; Klauda, Jeffery B; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Most molecular dynamics (MD) simulations of bacterial membranes simplify the membrane by composing it of only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or in some cases with1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) as well. However, an important constituent of bacterial membranes are lipids with a cyclopropane ring in the acyl chain. We developed a complex membrane that reflects the diverse population of lipids within E. coli cytoplasmic membranes, including cyclic lipids. Differences between the deuterium order profile of cyclic and monounsaturated lipids are observed. Furthermore, inclusion of the ring decreases the surface density of the bilayer and produces a more rigid membrane as compared to POPE/POPG membranes. Additionally, the diverse acyl chain length creates a thinner bilayer which better matches the hydrophobic thickness of E. coli transmembrane proteins. We believe the complex membrane is more accurate and suggest the use of it in MD simulations rather than simple membranes.Item Food Safety Implications of Biofilms Formed by Resident Bacteria in Fresh-cut Processing Environments(2013) Liu, Tong; Lo, Yangming M.; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Biofilms on equipment surfaces can be vectors for cross-contaminations in food processing facilities. A particular problem is that biofilms can protect pathogenic bacteria from daily cleaning and disinfection operations. In the present study, microflora were recovered from fresh-cut processing facilities, identified, and tested for biofilm forming potential. Subsequently, dual-species biofilms of selected isolates and Escherichia coli O157:H7 were investigated. Approximately 30% of the isolates were potent biofilm formers, producing large amounts of biomass. A hundred and seventeen tested isolates were identified into 23 genera, including plant related bacteria and coliforms with some opportunistic pathogens. Dual-species biofilms formed by Burkholderia caryophylli or Ralstonia insidiosa and E. coli O157:H7 manifested increased biomass in comparison to their monocultures. Additionally, about a one-log unit increase of E. coli O157:H7 cell counts were observed for both dual-species biofilms. To test the effects of environmental factors on growth of R. insidiosa and E. coli O157:H7 in dual-species biofilms, factors tested included low temperature (10 oC), media with different composition of nutrient sources (10% TSB, M9, 1.25% Cantaloupe Juice) and a continuous culture system with limited nutrients. E. coli O157:H7 cell counts increased for all tested conditions. To examine bacterial localization within biofilms, confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) were used. Images showed distinct spatial distributions with E. coli O157:H7 commonly located at the bottom and also interspersed among R. insidiosa cells. To test the interactions with other pathogens, R. insidiosa was co-cultured with Listeria monocytogenes, Salmonella spp., and Shiga toxin-producing E. coli. Cells counts for 7 out of 9 tested pathogenic bacteria strains were increased (0.36-1.60 CFU log units). It is notable that the biomass formed by R. insidiosa and L. monocytogenes was much greater than those produced by other tested combinations. These results indicate that R. insidiosa could be a food safety risk in fresh-cut processing environments by providing protective habitats for pathogenic bacteria.Item Comparing Source of Agricultural Contact Water and the Presence of Fecal Indicator Organisms on the Surface of 'Juliet' Grape Tomatoes(2010) Pahl, Donna Marie; Walsh, Christopher S; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Microbial water quality standards exist to prevent food safety outbreaks due to the use of agricultural water, although little is known about how the levels the fecal indicator organisms in water relate to the counts on the tomato fruit surface. This study used fecal indicator organisms commonly used in microbial water quality standards (Enterobacteriaceae, total coliforms, fecal coliforms, and E. coli) to monitor the water quality of surface ponds, a groundwater source, and the phyllosphere of treated grape tomatoes over two growing seasons. Water source and date caused a significant difference in the counts of fecal indicator organisms. Variability in bacterial counts was found in the surface water sources over the course of the season, partially explained by environmental variables such as water temperature, pH, precipitation, and air temperature. The microbial counts on the surfaces of the tomato fruit did not reflect the water treatments applied to the plants.